Project Summary The primary goal of this proposed project is to identify and characterize the human skeletal stem cell (hSSC) and the lineage restricted progenitors of bone, cartilage, and bone marrow stromal tissues that it generates. Building on our recent publication in Cell detailing the mouse SSC, our ultimate objective is to create a detailed lineage map of human skeletogenesis, as seen in hematopoiesis, with a mulitpotent stem cell generating various lineages in a niche that regulates differentiation. As demonstrated in our mouse study, we will achieve this by first purifying human skeletal stem and progenitor cells to a very high level of homogeneity (Aim 1) and then secondly by examining the transcriptional and translational expression of the cellular subsets, before finally defining and probing the inter-relationship between the stem and progenitor cells. These steps will, thus, enable us to strategize how to manipulate the SSC niche to drive fate determination of bone, cartilage or bone marrow stroma to affect clinical need (Aim 2). Our investigation of the mSSC niche regulation also led us to discover specific combinations of bone morphogenetic protein-2, Wnt and VEGF that could induce de novo formation of SSC even in non-skeletal (adipose) tissue. We will explore if similar morphogen combinations could also induce hSSC formation and de novo generation of bone, cartilage, or stroma from plentiful human adipose stromal populations (Aim 3). Despite the utility of mouse models, recent reports describe dramatic differences between mouse and human immunology, which has a direct impact on the development of novel therapeutics. Therefore, in order to truly affect clinical translation, it is prudent to first identify the hSSC to identify key genetic pathways that are conserved in mouse and human skeletogenesis and to reveal the genetic mechanisms underlying differences between mouse and humans. We are confident that the expertise we acquired upon implementation of our mSSC strategy puts us in a unique position to characterize human counterparts of the mouse skeletal stem and progenitor cell. Our new substantial preliminary human skeletal stem/progenitor data from both fetal and adult tissue strongly affirm the technical feasibility of our approach and the existence of the hSSC. The proposed experiments in this application, if supported, would clear the path to practical translation of stem cell regenerative medicine for skeletal diseases.